This project centers on identifying biomarkers of catecholaminergic denervation in autonomic synucleinopathies. We are examining whether biomarkers of catecholaminergic neurodegeneration predict PD in at-risk individuals. We have also noted particular patterns of cerebrospinal fluid (CSF) catechols that provide specific biomarkers of parkinsonism. Recently we have begun to examine whether immunofluorescence microscopy can be used to identify sympathetic noradrenergic denervation and the relationship of such denervation to alpha-synucleinopathy. (1) Cardiac sympathetic denervation predicts PD in at-risk individuals: By the time a person develops the motor manifestations of Parkinsons disease (PD), substantial loss of nigrostriatal dopamine neurons has already occurred. There is great interest in identifying biomarkers that can detect pre-clinical PD. Braaks neuropathological staging concept imputes early autonomic involvement. We completed subject accrual and analyzed 3-year follow-up data from a small prospective cohort study about the utility of neuroimaging evidence of cardiac sympathetic denervation in predicting PD among individuals with multiple PD risk factors. Subjects provided information about family history of PD, olfactory dysfunction, dream enactment behavior, and orthostatic hypotension at a protocol-specific website. From this pool, 27 people with at least 3 confirmed risk factors underwent cardiac 18F-dopamine positron emission tomographic scanning and were followed for at least 3 years. Interventricular septal and left ventricular free wall concentrations of 18F-dopamine-derived radioactivity were measured. Of the 27 subjects, 4 were diagnosed with PD within the 3-year follow-up period (Pre-Clinical PD group); 23 risk-matched (mean 3.2 risk factors) subjects remained disease-free (No-PD group). Compared to the No-PD group, the Pre-Clinical PD group had lower initial values for septal and free wall concentrations of 18F-dopamine-derived radioactivity (p=0.0248, 0.0129). All 4 Pre-Clinical PD subjects had evidence of decreased cardiac sympathetic innervation in the interventricular septum or left ventricular free wall, in contrast with 3 of 23 (13%) No-PD subjects (p=0.0020 by Fishers exact test). Thus, people with multiple PD risk factors and diagnosed with PD within 3 years have evidence of antecedent cardiac sympathetic denervation. The findings fit with Braaks staging concept.1 (2) Cerebrospinal fluid 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) predict PD: The PDRisk prospective cohort study is also testing whether individuals with multiple risk factors for PD and with CSF neurochemical biomarkers of central dopamine deficiency have pre-clinical PD. After confirmation of at least 3 risk factors as described above, the participants underwent inpatient testing including lumbar puncture for assays of CSF levels of DOPA (the precursor of dopamine) and DOPAC (the main neuronal metabolite of dopamine). CSF DOPA and DOPAC levels both distinguished the pre-clinical group from the group remaining free of PD (p=0.0302, p=0.0190). All 3 subjects with both low DOPA (<2.63 pmol/mL) and low DOPAC (<1.22 pmol/mL) levels developed clinical disease by 3 years, whereas none of 14 subjects with both normal DOPA and DOPAC levels did so (p=0.0015). Thus, in people with multiple PD risk factors, those with low DOPAC and DOPAC levels seem to have pre-clinical PD.2 (3) Elevated CSF ratios of cysteinyl-dopamine/3,4-dihydroxyphenylacetic acid in parkinsonian synucleinopathies: We published previously that PD and multiple system atrophy (MSA) both involve decreased CSF levels of DOPAC. In the cytoplasm, dopamine undergoes enzymatic oxidation to form DOPAC as well as spontaneous oxidation to form 5-S-cysteinyl-dopamine (Cys-DA). Our laboratorys unique capability to assay Cys-DA and DOPAC and simultaneously led to the discovery that in PD and parkinsonian MSA (MSA-P), CSF Cys-DA/DOPAC ratios averaged more than twice control, whereas in pure autonomic failure (PAF), a rare form of synucleinopathy that does not involve parkinsonism, the mean Cys-DA/DOPAC ratio was normal. Therefore, in synucleinopathies an elevated CSF Cys-DA/DOPAC ratio seems to provide a specific biomarker of parkinsonism. Oxidative stress or decreased activity of aldehyde dehydrogenase (ALDH) in the residual nigrostriatal dopaminergic neurons may explain the increased CSF Cys-DA/DOPAC ratios found in parkinsonian synucleinopathies (unpublished observations). (3) Autoimmunity-associated autonomic failure with sympathetic denervation: There is increasing evidence that in rare cases, pandysautonomia reflects autoimmune attack. We reported a case of autoimmunity-associated autonomic failure in a young adult woman who developed arthritis followed 3 years later by pandysautonomia. There was early recovery of parasympathetic functions but persistent neurogenic orthostatic hypotension from post-ganglionic sympathetic denervation. Clinical laboratory testing indicated variable amounts of sympathetic neuronal re-sprouting in the heart, kidneys, eyes, and body as a whole upon follow-up evaluation after 1.5 years.3 (4) Pure autonomic failure without synucleinopathy: Pure autonomic failure (PAF) is an uncommon form of chronic autonomic failure manifesting with neurogenic orthostatic hypotension and evidence of sympathetic noradrenergic denervation unaccompanied by signs of central neurodegeneration. It has been proposed that PAF is in the family of Lewy body diseases such as PD, characterized by intra-neuronal deposition of the protein alpha-synuclein in Lewy bodies and neurites. A middle-aged man with previously diagnosed PAF experienced a sudden, fatal cardiac arrest. He was autopsied, and tissues were harvested for neurochemical and immunofluorescence studies. Post-mortem microscopic neuropathology showed no Lewy bodies, Lewy neurites, or alpha-synuclein deposition by immunohistochemistry anywhere in the brain of periphery. The patient had markedly decreased immunofluorescent tyrosine hydroxylase in sympathetic ganglion tissue without detectable alpha-synuclein even in rare residual nests of tyrosine hydroxylase-containing ganglionic fibers. Thus, in PAF sympathetic noradrenergic denervation can occur without concurrent Lewy bodies or alpha-synuclein deposition in the brain or sympathetic ganglion tissue.4 REFERENCES 1. Goldstein DS, Holmes C, Lopez G, Wu, Tianxia, Sharabi Y. Cardiac sympathetic denervation predicts PD in at-risk individuals. Parkinsonism Relat Disord (submitted). 2. Goldstein DS, Holmes C, Lopez G, Wu T, Sharabi Y. Cerebrospinal fluid dihydroxyphenylalanine and dihydroxyphenylacetic acid predict Parkinson disease. Mov Disord (submitted). 3. Goldstein DS, Holmes C, Sullivan P, et al. Autoimmunity-associated autonomic failure with sympathetic denervation. Clin Auton Res 2016. 4. Isonaka R, Holmes C, Cook GA, Sullivan P, Sharabi Y, Goldstein DS. Pure autonomic failure without synucleinopathy. Clin Auton Res 2017.